Multidentate arrays

ABSTRACT

A method of evaluating for the presence of a target polynucleotide in a sample, using an addressable array of multiple polynucleotide probes linked to a substrate. The sample is exposed to the array and a set of polynucleotide target probes, such that target polynucleotide which may be present will bind to a predetermined feature of the array through multiple target probes of the set by forming at respective target regions on a target molecule, simultaneous hybrids with anti-target regions of the multiple target probes. A binding pattern on the array is observed and the presence of the target polynucleotide evaluated based on the observed binding pattern. Kits using such arrays, and methods for selecting target probes are further provided.

FIELD OF THE INVENTION

This invention relates to arrays, particularly biopolymer arrays suchpolynucleotide arrays, which are useful in diagnostic, screening, geneexpression analysis, and other applications.

BACKGROUND OF THE INVENTION

Arrays of biopolymers, such as arrays of peptides or polynucleotides(such as DNA or RNA), are known and are used, for example, as diagnosticor screening tools. Such arrays include regions (sometimes referenced asfeatures or spots) of usually different sequence biopolymers arranged ina predetermined configuration on a substrate. The arrays, when exposedto a sample, will exhibit a pattern of binding which is indicative ofthe presence and/or concentration of one or more components of thesample, such as an antigen in the case of a peptide array or apolynucleotide of particular sequence in the case of a polynucleotidearray. The binding pattern can be detected, for example, by labeling allpotential targets (for example, DNA) in the sample with a suitable label(such as a fluorescent compound), and accurately observing thefluorescence pattern on the array.

In one application, arrays of oligonucleotide probes provide usefultools for simultaneous evaluation of the levels of expression of largesets of genes (“expression profiling”). The probe arrays used inexpression profiling can be produced in two ways: (i) oligonucleotideprobes can be synthesized in situ on the array surface, usinglocation-addressable adaptations of phosphoramidite chemistry (forexample, photo-deprotection, or printing of phosphoramidites using aninkjet type printer); (ii) whole oligonucleotide probes synthesizedeither by phosphoramidite chemistry or enzymatic methods (for example,PCR) can be deposited on a surface designed to form either a strongnon-covalent attachment to DNA (for example, poly-L-lysine) or acovalent attachment to a chemically unique group added to theoligonucleotide during synthesis (for example, a modified basecontaining a primary aliphatic amine). Noncovalent attachment may besubsequently turned into covalent attachment by methods such as UVphoto-cross linking. Chemical synthesis is used to produce probesshorter than 50 nucleotides, while enzymatic methods are used to producelonger probes (100-1000 nucleotides).

Synthetic nucleotide probes (either synthesized in situ or depositedwhole) can potentially discriminate between closely related mRNA's,because they can be designed to probe the most different portions of thetarget sequences, and because the effects of these differences areproportionately greater for shorter probes. This ability is important,because many genes in higher organisms are members of families ofrelated genes. However, shorter probes suffer from the difficulty thatthey do not associate with their targets as strongly as longer probes(that is, they have a lower binding constant or binding affinity). Thisweaker association makes it very difficult to produce oligonucleotideprobes that can unequivocally detect concentrations lower than about 0.1pM for the best cases, and typical detection limits are in the range of1 pM-10 pM. This results in a sensitivity gap. For example, if all ofthe mRNA in a sample of 10⁶ cells (a typical size for sampling aprecious specimen, such as a biopsy sample) is converted into labeledcDNA, and the resulting material is resuspended in a volume of 100 μl,then the final concentration of the cDNA derived from a message presentat 1 copy per cell is$\frac{( {10^{6}\quad {cells}} )( \frac{1{copy}}{cell} )}{( {6.02 \times 10^{23}\frac{copies}{mole}} )( {10^{- 4}\quad {liters}} )} = {{1.66 \times 10^{14}\quad M} = {0.017\quad {pM}}}$

This is a factor of 5 lower than the lowest limit of detection achievedwith current oligonucleotide probe - polynucleotide target combinations,and a factor of 50-500 below more typical detection limits.

The sensitivity gap can be closed by employing a target amplificationscheme, such as linear amplification by RNA transcription or asymmetricPCR. However, this adds both complication and cost to the assay. Inaddition, losses of mRNA during sample preparation, amplification,inhibition by sample-derived impurities and problems with probespecificity (which necessitate more stringent conditions and lowersignal levels) can together use up most or all of the sensitivity marginprovided by target amplification. Finally, lowering the number of cellsrequired per sample would greatly improve the applicability of arrays,since it would then be possible to perform entire array analyses onsamples provided by microsampling methods, such as needle biopsy andlaser-assisted micro-dissection.

Arrays which utilize longer probes can exhibit binding constants highenough to yield detection limits in the 10⁻¹⁵ M range. However, thisimproved performance comes at the costs of lost specificity within genefamilies and loss of the ability of design probes to hybridize to themost unique target subsequences.

Solutions to the sensitivity gap from using synthetic oligonucleotideprobes, include target amplification, signal amplification, the use ofhigh sensitivity labels and the use of modified probe nucleotidechemistries. Target amplification, described in the previous section, isa well established method for overcoming an intrinsic binding constantthat is too low. It solves the problem directly, by increasing theamount of target by a well-controlled factor that is relativelyindependent of the target sequence. The disadvantages of targetamplification are the complication and cost added to sample preparation.Another solution is signal amplification, which is achieved bymultiplying the number of detectable labels attached to a given targetmolecule that binds to an array feature. Many sample labeling schemesincorporate a basic form of signal amplification by the simple expedientof attaching the label (for example, a fluorophore) to one or more ofthe nucleotide triphosphates used by the transcription-based system thatproduces labeled target oligonucleotide. More elaborate schemes, such asbinding of labeled biotin-streptavidin complexes and the formation ofsandwiches between surface-bound probes, unlabeled targets and highlylabeled second probes (for example, branched DNA probes) have also beenemployed. These methods, like target amplification, are relativelycostly and complicated and further rely on the binding of a very smallnumber of molecules. This can result in an added source of noise derivedfrom the probabilistic binding of small numbers of target molecules.High sensitivity labels (for example, radioisotopes, chemiluminescentlabels) are a special case of signal amplification. The main advantageof such methods is that they generate signal against a very lowintrinsic background. The disadvantage is that these labels are not asconvenient or safe as fluorescent probes. In addition, radioisotopesprovide lower spatial resolution than optical probes.

Probes that incorporate modified bases or backbones into polynucleotidesmay be capable of providing much higher per base binding free energiesthan conventional DNA probes. The main disadvantages of this approachare the relatively poor state of development of synthetic schemes forproducing probes that incorporate nucleotide analogues and therelatively poor state of characterization of the benefits derived fromthe use of such alternate chemistries. At present most of theperformance enhancement available from modified polynucleotidechemistries is theoretical.

U.S. Pat. No. 4,731,325 describes an arrangement using two or threeidentifying nucleic acid fragments homologous to a nucleic acid to beidentified. The patent states that if simultaneous identification ofseveral different nucleic acids is desired, it is necessary to useseparate filters to which are attached the required fragments. A paperby Gentalen et al., “A novel method for determining linkage between DNAsequences: hybridization to paired probe arrays” Nucleic Acids Research,1999, Vol. 27, No. 6 1485-1491 describes co-operative hybridization toestablish physical linkage between two loci on a DNA strand. Thesereference, and all other references cited in this application, areincorporated in this application by reference. However, cited referencesor art are not admitted to be prior art to this application.

It would be desirable then, to provide a means for detecting a targetusing probes, particularly in the form of an addressable array, whichcan provide good binding affinity for the target. It would also bedesirable that any such means be relatively simple to fabricate. Itwould further be desirable that a means be provided for aiding in theselection of such probes.

SUMMARY OF THE INVENTION

The present invention then, provides for high affinity of probes to atarget by using an array feature in which two or more probes are presentwhich together bind with respective regions of a target at two or moreregions (that is, the feature exhibits “multidentate” binding”).Furthermore, the present invention appreciates that in the context ofsuch systems, due to secondary structure of target polynucleotide inparticular, finding a good set of target probes is not necessarily amatter of simply selecting probe sequences complementary to targetsequences of the target polynucleotide. The invention then, alsoprovides for a means aiding in the selection of probes suitable toprovide the foregoing multidentate binding.

In one aspect, the present invention provides a method of evaluating forthe presence of a target polynucleotide in a sample, using anaddressable array of multiple polynucleotide probes linked to asubstrate. The sample is exposed to the array and a set ofpolynucleotide target probes, such that target polynucleotide which maybe present will bind to a predetermined feature of the array throughmultiple target probes of the set. This occurs by respective targetregions on a target molecule, forming simultaneous hybrids withanti-target regions of the target probes. It will be appreciated, ofcourse, that the target probe set is either bound to the substrateeither before, during, or after exposure to the substrate. For example,the target probe can be either directly bound to the substrate (forexample, by linking to the substrate before the sample is exposed to thearray), or indirectly bound to the substrate (such as through a captureprobe). Thus, individual target molecules will be bound to the array atmultiple locations along the molecule (sometimes referenced herein as“multidentate binding”).

Optionally, a binding pattern on the array may then be observed and thepresence of the target polynucleotide evaluated based on the observedbinding pattern.

However, this can be done either within a short time following theforegoing steps, or potentially at some indefinite later time.

The method of the invention also allows for the presence of multipledifferent target polynucleotides to be evaluated. In this mode, thesample is exposed to multiple different sets of target probes such thateach of the different polynucleotide molecules which may be present willbind to a corresponding predetermined features of the array throughmultiple target probes of a corresponding set by forming at respectivetarget regions, simultaneous hybrids with the multiple probes of thecorresponding set. Thus, each particular type of target molecule will bebound to the corresponding feature of the array at multiple locationsalong the molecule. Note however, throughout this invention a giventarget polynucleotide can in fact be a class of polynucleotides in whichno discrimination is required between individual members of the class.In such a case, a member of the class may have target regions which arethe same or similar in sequence to other members of the class.

In an aspect of the invention where the target probes are to beindirectly bound to the substrate, the target probes also includeanti-capture regions. Also, the predetermined feature of the arrayincludes a set of capture probes linked to the substrate which havecapture regions which will hybridize with the anti-capture regions. Inthis manner, multiple molecules of a given type of target polynucleotidewhich may be present will each indirectly bind to the predeterminedfeature through the corresponding set of target probes, by theanti-target regions of target probes forming the simultaneous hybridswith the respective target regions and by the anti-capture regionshybridizing with the capture regions of the capture probes.Alternatively, the capture probes may not be linked to the substrate, inwhich case the predetermined region of the substrate should be providedwith a further probe to bind to some region of the capture probe (thatis, the target would become bound to the substrate at least through aset of target probes, a set of capture probes, and some other probes atthe predetermined feature on the array).

Note that in the invention, the target regions of any one targetpolynucleotide (that is, any one type of target polynucleotide) could beof the same sequence (in which case, the members of the set of targetprobes can be the same, that is the “set” corresponding to that targetpolynucleotide has only one member in the form of one type of targetprobe) or of different sequence (in which case the anti-target regionsof the multiple target probes of the set may also be of differentsequence to hybridize with the different sequenced target regions thusthe “set” has more than one member in the form of multiple types oftarget probes). For example, target regions of a given polynucleotidemay differ from one another by at least two (or three, or four)nucleotides, while the anti-target regions of the corresponding probeset also differ from one another by at least two (or three, or four)nucleotides. However, it will be appreciated that when there is morethan one target polynucleotide (that is more than one type of targetpolynucleotide), target regions of one target polynucleotide should beof different sequence from those of another target polynucleotide sothat one does not bind to the array feature intended for the other. Inthe case of indirect binding of target to the array using captureprobes, anti-target regions within a given target probe set may also bethe same or different. Also, the anti-capture regions within a givenprobe set may be the same or different. It will also be appreciated inthe present method that any target nucleotide may be determined to bepresent (positive test) or not present (negative test) based on theobserved 25 binding pattern.

The present invention further provides an apparatus which can be used inmethods of the present invention to evaluate for the presence of atarget polynucleotide in a sample. Such an apparatus includes anaddressable array of multiple polynucleotide probes linked to thesubstrate. The apparatus further includes a set of polynucleotide targetprobes which may or may not be linked to the substrate as part of thearray. By this arrangement, target polynucleotide which may be presentin a sample exposed to the array will bind to a predetermined feature ofthe array through multiple target probes of the set by forming atrespective target regions on a target molecule, simultaneous hybridswith anti-target regions of the multiple target probes. Again, thetarget regions may be of the same or different sequence. In the casewhere the target regions are of different sequence, the set of targetprobes has at least two target probes with different sequenceanti-target regions. For ease of reference, an apparatus for evaluatingthe presence of a target polynucleotide will sometimes be referred to inthis application as a “kit” to distinguish from the apparatus of thepresent invention for evaluating target probes. However, it will beappreciated that such a “kit” may be simply an array (and hence, an“array of the present invention” is also referenced).

In one aspect, where the presence of multiple different targetpolynucleotides are to be evaluated by the apparatus, the apparatusincludes multiple different sets of target probes. In this manner thedifferent polynucleotide molecules which may be present in the samplewill bind to respective different predetermined features of the arraythrough multiple target probes of respective sets, by forming atrespective target regions, simultaneous hybrids with the multiple probesof the respective sets.

In a particular aspect, the anti-target regions of the multiple targetprobes of all the sets may be of different sequence. This isdistinguished from the case where anti-target regions of target probeswithin a given set may be the same, where the target regions of thetarget corresponding to that set are of the same sequence. In this casethe designer of target probes may wish to specifically select candidateprobes which will bind to such repeated regions in order to promotemultidentate binding. Also, the apparatus may include target probeslinked to the predetermined array feature (for direct target binding) ormay additionally include target probes not linked to the substrate (forindirect target binding), both as described above. Similarly, theapparatus may include an array, and probe sets suitable for carrying outany of the methods of the present invention.

The present invention further provides a method of evaluatingpolynucleotide target probes on their ability to form simultaneoushybrids with respective regions of a same target polynucleotidemolecule. The method includes selecting candidate probes which canpotentially hybridize with selected respective candidate target regionsof the target polynucleotide, based on the sequence of the selectedcandidate target regions. While the candidate probes will typically beexact complements of the selected candidate target regions, they neednot necessarily be so. The candidate probes are tested on their abilityto actually hybridize individually with respective candidate targetregions. At least two of the candidate probes are further selected,which actually hybridized individually with at least a predeterminedefficiency with respective candidate target regions. The furtherselected candidate probes are tested on their ability to formsimultaneous hybrids with the respective candidate target regions.Optionally, multiple different relative concentrations of the furtherselected candidate probes may be tested in the foregoing manner.

In a particular aspect of the evaluation method, the target probes areevaluated on their ability to form the simultaneous hybrids, when theprobes are linked to a substrate (such as that of a polynucleotidearray). In this case, at least the further selected candidate probes aretested on their ability to form simultaneous hybrids, when linked to thesame substrate. Further, the initially selected candidate probes mayalso be linked to a substrate when being tested on their ability toactually hybridize individually with respective candidate targetregions. In either case, the initially or further selected candidateprobes may be linked to the same substrate in the form of an addressablearray.

In the evaluation method of the present invention, the selectedrespective candidate target regions may be spaced apart along the targetpolynucleotide (such as at regular or irregular intervals, or based uponregions which from other analysis are thought to be particularly goodtarget regions). The method may additionally include selecting at leastone additional candidate target region based on a hybridization pattern(such as the efficiency of binding) of those candidate probes tested ontheir ability to actually hybridize individually, and repeating theselecting and testing candidate probes to hybridize individually, forthe additional candidate target region. Additional iterations of thesesteps can optionally be repeated as often as desired.

The present invention further provides a method of fabricating anaddressable array of multiple polynucleotide probes linked to asubstrate. In this method, target probes are evaluated according to themethod above. Further selected candidate probes which actually formedsimultaneous hybrids with the respective candidate target regions withat least a predetermined efficiency, are linked to the substrate at apredetermined feature.

The present invention further provides an apparatus comprising acomputer for evaluating polynucleotide target probes on their ability toform simultaneous hybrids with respective regions of a same targetpolynucleotide molecule. In one aspect, the apparatus executes at leastthe steps of: (a) selecting candidate probes which can potentiallyhybridize with selected respective candidate target regions of thetarget polynucleotide, based on the sequence of the selected candidatetarget regions; (b) receiving results of testing candidate probes ontheir ability to actually hybridize individually with respectivecandidate target regions; (c) further selecting at least two of thecandidate probes which actually hybridized individually with at least apredetermined efficiency with respective candidate target regions. Inanother aspect, the executed steps include the foregoing steps (a) and(b) and a step (c) in which at least one additional candidate targetregion based on the results received in step (b) is selected, and atleast step (a) repeated for the additional candidate target region. In astill further aspect, the computer may additionally receive results oftesting the further selected candidate probes on their ability tosimultaneously hybridize with respective target regions of the targetpolynucleotide. These results can optionally include the testing of thefurther selected candidate probes on their ability, at multipledifferent relative concentrations, to simultaneously hybridize withrespective target regions of the target polynucleotide. In a stillfurther particular aspect, these results at multiple differentconcentrations can be analyzed and a relative concentration of themembers of the set of such candidate probes selected, and optionallylinked to a substrate at a predetermined feature (typically along withother probes at their respective features) to produce an array of thepresent invention.

A computer program product is further provided by the present invention,which includes a computer readable storage medium having a computerprogram stored on the medium for performing, when loaded into acomputer, at least the steps of any of the methods or apparatus of thepresent invention.

It will be appreciated that while in the above description reference ismade to polynucleotides, the present invention can be extended to othertarget moieties and suitable probes, for example, to biopolymers otherthan polynucleotides, such as peptides (which is used here to includeproteins). Thus, a description of a broader aspect of the presentinvention may be obtained simply by deleting “polynucleotide” in thedescription of the invention, and replacing “hybridizing” or similarterms with simply “binding”, and “hybrids” with “binding partners”. Inone aspect, “polynucleotide” can be replaced with “biopolymer”, or in aparticular aspect with “peptide”.

The various aspects of the present invention can provide any one or moreof a number of useful benefits. For example, a means is provided fordetecting a target using probes, particularly in the form of anaddressable array, which can provide good binding affinity for thetarget. Such means is relatively simple to fabricate. A means is furtherprovided for aiding in the selection of such probes.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described with reference to thedrawings in which:

FIG. 1 illustrates a chip carrying multiple arrays, at least one ofwhich is of the present invention, wherein target probes are directlylinked to the substrate;

FIG. 2 is an enlarged view of a portion of FIG. 1 showing multiple spotsor regions of one array;

FIG. 3 is an enlarged illustration of a portion of the substrate of FIG.1;

FIG. 4 is an enlarged illustration of a portion of another array and amethod of the present invention, wherein target probes are directlylinked to the substrate;

FIG. 5 illustrates an array and method of the present invention whereintarget probes are indirectly linked to a substrate;

FIG. 6 is a schematic of an apparatus of the present invention and theuse of such an apparatus in a probe evaluation method of the presentinvention;

FIG. 7 is a flowchart illustrating a method of the present invention,including the steps executed by a computer program carried by a computerprogram medium of the present invention;

FIG. 8 illustrates a kit of the present invention; and

FIG. 9 is a graph illustrating experimentally determined bindingefficiency of candidate probes to a target region, versus position ofthe target region along a target polynucleotide, as discussed further inthe Example below.

To facilitate understanding, identical reference numerals have beenused, where practical, to designate similar elements that are common tothe figures.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present application, unless a contrary intention appears,the terms following terms refer to the indicated characteristics. A“biopolymer” is a polymer of one or more types of repeating units.Biopolymers are found in biological systems and particularly includepeptides or polynucleotides, as well as such compounds composed of orcontaining amino acid or nucleotide analogs or non-nucleotide groups.This includes polynucleotides in which the conventional backbone hasbeen replaced with a non-naturally occurring or synthetic backbone, andnucleic acids in which one or more of the conventional bases has beenreplaced with a synthetic base capable of participating in Watson-Cricktype hydrogen bonding interactions. Polynucleotides include single ormultiple stranded configurations, where one or more of the strands mayor may not be completely aligned with another. While probes and targetsof the present invention will typically be single-stranded, this is notessential. A “nucleotide” refers to a sub-unit of a nucleic acid and hasa phosphate group, a 5 carbon sugar and a nitrogen containing base, aswell as analogs of such sub-units. Specifically, a “biopolymer” includesDNA (including cDNA), RNA and oligonucleotides, regardless of thesource. An “oligonucleotide” generally refers to a nucleotide multimerof about 10 to 100 nucleotides in length, while a “polynucleotide”includes a nucleotide multimer having any number of nucleotides. A“biomonomer” references a single unit, which can be linked with the sameor other biomonomers to form a biopolymer (for example, a single aminoacid or nucleotide with two linking groups one or both of which may haveremovable protecting groups). A biomonomer fluid or biopolymer fluidreference a liquid containing either a biomonomer or biopolymer,respectively (typically in solution). An “array”, unless a contraryintention appears, includes any one or two dimensional arrangement ofaddressable regions bearing particular biopolymer moieties (for example,different polynucleotide sequences) associated with that region. Anarray is “addressable” in that it has multiple regions of differentmoieties (for example, different sequences) such that a region at aparticular predetermined location (an “address”) on the array (a“feature” of the array) will detect a particular target or class oftargets (although a feature may incidentally detect non-targets of thatfeature). In the present case, the polynucleotide (or other) target willbe in a mobile phase (typically fluid), while probes for the target(“target probes”) may or may not be mobile (as described in thisapplication). “Hybridizing” and “binding”, with respect topolynucleotides, are used interchangeably. “Binding efficiency” refersto the productivity of a binding reaction, measured as either theabsolute or relative yield of binding product formed under a given setof conditions in a given amount of time. “Hybridization efficiency” is aparticular sub-class of binding efficiency, and refers to bindingefficiency in the case where the binding components are polynucleotides.It will also be appreciated that throughout the present application,that words such as “upper”, “lower” are used in a relative sense only. A“set” may have one type of member or multiple different types. “Fluid”is used herein to reference a liquid. By one item being “remote” fromanother is referenced that they are at least in different buildings, andmay be at least one, at least ten, or at least one hundred miles apart.Reference to a singular item, includes the possibility that there areplural of the same items present.

Referring first to FIGS. 1-3, typically kits and methods of the presentinvention use a contiguous planar substrate 10 carrying multiple arrays12 disposed across a first surface 1 a of substrate 10 and separated byareas 13. The arrays on substrate 10 can be designed for testing asample or for evaluating probes on their ability to form hybrids. Whileten arrays 12 are shown in FIG. 1 and the different embodimentsdescribed below may use substrates with particular numbers of arrays, itwill be understood that substrate 10 and the embodiments to be used withit, may use any number of desired arrays 12. Similarly, substrate 10 maybe of any shape, and any apparatus used with it adapted accordingly.Depending upon intended use, any or all of arrays 12 may be the same ordifferent from one another and each will contain multiple spots orfeatures 16 of biopolymers in the form of polynucleotides. A typicalarray may contain from 100 to 100,000 regions. All of the features 16may be different, or some or all could be the same. Each feature carriesa predetermined polynucleotide having a particular sequence, or apredetermined mixture of polynucleotides. It will be appreciated though,that there need not be any space separating arrays 12 from one another,nor features 16 within an array from one another.

FIG. 3 particularly illustrates an addressable array of multiple sets ofpolynucleotide probes linked to substrate 10 and forming part of thearray, wherein features 16 (specifically, each of features 16 a, 16 b,and 16 c) are shown as each carrying sets of polynucleotide targetprobes. Such an array is useful in kits of the present invention forevaluating for the presence of multiple different polynucleotides, or intesting the ability of multiple different candidate probes of a set tosimultaneously hybridize with respective target features of a particulartarget, as will be described further below. Specifically, feature 16 acarries a first set of polynucleotide target probes or candidate targetprobes, which set has two members of different sequence, namely probes20, 22. Similarly, feature 16 b carries a second set of polynucleotidetarget or candidate target probes 24, 26, respectively, while feature 16c carries a third set of target or candidate target probes 28, 30,respectively. Thus each feature 16, as illustrated in FIG. 3, can bindto two target features. Typically, the entire sequence of each probe 20,22, 24, 26, 28, 30 will serve as the anti-target region. It will beappreciated that FIG. 3 is not to scale and that, in particular, each ofthe features 16 will have many more polynucleotide molecules of each setthan illustrated.

The array of FIGS. 1-3 can be used to evaluate for the presence ofmultiple polynucleotides in a sample by exposing the sample to the arrayunder hybridizing conditions (that is, conditions which allow targetsequences to hybridize to corresponding anti-target sequences). Thus,the sample is exposed to multiple different sets of target probes. Itwill be assumed for this example, that the entire sequences of thetarget probes in FIGS. 1-3 act as anti-target sequences. Spots 16 a, 16b, 16 c will allow for the evaluation of the presence of three differentpolynucleotide targets. In particular, a first target polynucleotidehaving two different sequence target regions sufficiently complementary(often, but not necessarily, exactly complementary) to respective targetprobes 20, 22 of the first set, when present will bind to feature 16 athrough the multiple target probes 20, 22 by forming at respectivetarget regions on a given target molecule, simultaneous hybrids with thea probe 20 and a probe 22. By forming “simultaneous hybrids” throughoutthis application does not imply any particular order of formation, butonly that the hybrids exist at the same time. Thus, the first target isbound to substrate 10 at its two target regions. Similarly, a secondtarget polynucleotide which may be present, will bind to feature 16 bthrough two target regions and complementary respective probes 24, 26.Likewise, a third target polynucleotide which may be present, will bindto feature 16 c through two target regions and complementary respectiveprobes 28, 30. Similarly, other features 16 can bind to respectivetargets although, of course, other features need not bind targets at tworegions (but could bind at just one, three, or another number ofregions). The resulting binding pattern (which includes the possibilityof no binding on the array), can be observed (such as by detection offluorescence in the case of fluorescently labeled targetpolynucleotides) and conclusions drawn as to the presence or absence ofthe different targets in the sample and optionally, of theirconcentrations (whether relative or absolute).

If desired, the array particularly illustrated in FIG. 3 can befabricated so that one or more target probe sets can each formsimultaneous hybrids with three or more target regions on acorresponding target probe. This can be accomplished by providing one ofthe target probe sets at a predetermined feature, with at least threepolynucleotide target probes with different sequence anti-targetregions. Where the entire length of a target probe is used as theanti-target region, as is typical in FIG. 3, this means that a givearray feature will carry at least three different sequence anti-targetprobes. A portion of an array with a set of three probes having ofdifferent anti-target sequence, is illustrated in FIG. 4. In FIG. 4 theillustrated portion of the array has an feature 16 d with two a firstset of target probes 20, 22 with different sequence anti-target regions21 and 23, respectively. Note that in this embodiment, only a portion ofthe illustrated target probes serve as anti-target regions. However, itwill be appreciated as mentioned above, that the entire target probesequence may serve as the anti-target sequence. The array further has anfeature 16 e with a second set of target probes 30, 32, 34 withrespective anti-target regions 31, 33, and 35.

When an array having the features 16 d, 16 e of FIG. 4 is exposed to asample to evaluate for the presence of target polynucleotides 34 and 40,target polynucleotide 34 which may be present will bind to feature 16 dby forming at respective two target regions 36 and 38, simultaneoushybrids with anti-target regions 21 and 23, respectively. Similarly,target polynucleotide 40 which may be present will bind to feature 16 eby forming at three target regions 42, 44, 46, simultaneous hybrids withrespective anti-target regions 31, 33 and 35, as illustrated in FIG. 4.Again the resulting binding pattern can be observed and the presence ofpolynucleotides 34, 40 evaluated based on the observed binding pattern.

Arrays with features such as those illustrated in FIGS. 3 or 4 (withmixed probe features), can be produced by mixing two or morepre-synthesized probes (as required by each probe set), then spottingthe mixture onto a surface designed to bind the mixture by covalentlinkage to a functional group not normally present in oligonucleotides(for example, a primary aliphatic amine or a sulfhydryl group) or bynon-covalent absorption and subsequent chemical or photochemicalcross-linking. Alternatively, the features can be synthesized in situ onthe substrate using phosphoramidite units with two distinct protectionchemistries and an ink jet type dispenser. Techniques using differentprotection chemistries are known. The resulting feature will display arandom microscopic mosaic of the probes in the original mixture. A giventarget molecule will then be capable of binding (by forming simultaneoushybrids) with the multiple, different probes of the corresponding probeset, as described above. Note that each binding targets a differenttarget sequence of the target polynucleotide. If the targetpolynucleotide is sufficiently flexible, or the target probes aresufficiently numerous, the binding events will take place independently,and the overall binding constant will be the product of the individualbinding constants. Thus, such multidentate features are capable ofdetecting lower concentrations of a target polynucleotide than would acorresponding monodentate feature (that is, a feature which only bindsto a target polynucleotide at a single region) carrying only one of thetarget probes of a multidentate feature.

The features illustrated in FIGS. 3 and 4 have the target probesdirectly bound to substrate 10. However, as mentioned above, in anotheraspect of the invention the target probes can be indirectly bound to thesubstrate. Such a configuration is similar to the directly boundconfigurations described above except that polynucleotide capture probesare bound to the substrate, and each target probe of a capture probe setincludes both an anti-target region and an anti-capture region which canbind to capture probes in turn bound to the substrate at a correspondingarray feature. Thus, the target probes act as intermediate or “bridgingprobes” between capture probes and target regions. This configurationcan be understood with reference to FIG. 5 in particular and isdiscussed below.

In particular, let X denote a surface bound probe sequence (a captureregion) at a predetermined array feature and which is unrelated to anytarget regions of interest. Let {overscore (X)} (an anti-capture regionon a target probe) denote the sequence exactly complementary to X.Further, let S¹, S², and the like denote different target probesequences (anti-target regions) of a target probe set, directed againstexactly complementary target regions (denoted {overscore (S₁)},{overscore (S₂)} and the like) in some target polynucleotide. Let Idenote a general intervening sequence in any target probe between ananti-capture region ({overscore (X)}), and the anti-target region({overscore (S₁)}, {overscore (S₂)}, and the like) of the same probe.Finally, let {overscore (T₁)}, {overscore (T₂)}, and the like, denotesequences in the target polynucleotide that are not complementary toprobe sequences (and do not hybridize therewith to any substantialextent). In this notation then, the target polynucleotide 50 would bewritten as

T ₁- {overscore (S₁)}-T ₂- {overscore (S₂)}-T ₃,

while first and second probes of a target probe set would be written as:

{overscore (X)}-I- S ₁ and {overscore (X)}-I- S ₂.

Kits of the present invention which use indirect binding of a targetprobe to the substrate then, would include an addressable array with asubstrate 10 (designated as a “surface”) in FIG. 5 having apredetermined feature at which are linked to substrate 10 capture probesX, as illustrated in FIG. 5. Other features (not shown in FIG. 5) on thearray which may be intended for evaluating the presence of other targetpolynucleotides, may use respective other capture probes and/or targetprobes as described herein. The kit further includes a set of targetprobes {overscore (X)}-I-S₁ and {overscore (X)}-I-S₂, and possibly otherdifferent sets of target probes for other array features intended forother target polynucleotides. While it is preferable that theanti-target regions are of different sequence, they could possibly bethe same (for example, both could be S₁) where a target polynucleotidehas two spaced apart target regions of the same sequence. Also, while itis simplest and therefore preferable that the capture regions X at thepredetermined feature are all the same, it is possible to use captureprobes at the predetermined feature which have different sequencecapture regions (such as X₁ and X₂) in which case different anti-capturesequences may be used (such as {overscore (X)}₁ and {overscore (X)}₂).

The use of a foregoing type of kit is also illustrated in FIG. 5. InFIG. 5, anti-target-target sequence duplex (hybrid) formation be denotedby ⇄. When a sample containing a target 50 is exposed to an array andtarget probes of the foregoing kit, under hybridizing conditions, thehybridization complex illustrated in FIG. 5 will form. That is, each ofmultiple molecules of target polynucleotide 50 will indirectly bind tothe predetermined feature of the array to which is linked capture probesX. This occurs by anti-target regions S₁ and S₂ of the multiple targetprobes {overscore (X)}-I-S₁ and {overscore (X)}-I-S₂ formingsimultaneous hybrids with target regions {overscore (S₁)} and {overscore(S₂)}, respectively, and by the anti-capture regions {overscore (X)}hybridizing with the capture regions of the capture probes X. Thisresults in bidentate binding (that is, two points of attachment) of thetarget, via the target probes. The foregoing complex of this sort can beassembled in multiple steps, by hybridizing an array with a featurehaving linked capture probe X, to the set of target probes and then totarget. In this case, the first-layer (bridging probes hybridized to thearray) can be stabilized by UV photo-crosslinking or chemicalcrosslinking. Alternatively, the hybridization complex can be assembledby mixing the target and target probes together, then hybridizing themixture to the array (either after the foregoing target/target probemixing, or simultaneously with that mixing).

A kit of the present invention may carry an array on substrate 10, whichhas at least one feature as described in connection with FIG. 5. The kitfurther includes a container 300 with the set of target probes(typically mixed together) of FIG. 5. The mixture in container 300 mayalso include other sets of intermediate target probes, as well asreference polynucleotides (used, for example, to confirm hybridizationconditions) or polynucleotide target probes in a preselectedconcentration but labeled differently from sample target probes of thesame sequence target regions (used as references to determine relativeamount of target sequence in a sample). All of the foregoing can beplaced in a single package 410 along with instructions printed on amedium 400 (for example, paper). Alternatively, for kits with arrays inwhich the target probes are directly linked to substrate 10 as in FIGS.3 and 4, the target probes need not be present in container 300 andfurther, if other types of mentioned probes are not required, container300 can be dispensed with altogether.

Finding a set of suitable target probes (whether directly or indirectlybound to a substrate) can be an time consuming, since for any giventarget polynucleotide there can be a large number of potential sequenceswhich can individually bind with all possible sub-sequences of a targetpolynucleotide. To then try all combinations of such possible potentialsequences (for example, all possible combinations of two such sequencesin the case where the target probe set has two members) becomes anunduly burdensome task. However, as mentioned above, the presentinvention provides an apparatus, and a method which can be executed bysuch an apparatus, for choosing good polynucleotide target probes for atarget probe set of any of the types described above. One such apparatusis illustrated in FIG. 6, and includes a computer 100 having an operatordisplay 104 and operator input device 106 (for example, a keyboardand/or mouse or other user operable pointing device). Computer 100includes a programmable processor as well as a drive for loading anevaluation program from a computer program product in the form of acomputer readable portable medium 110 (which may, for example, be amagnetic or optical disk or tape). The computer readable storage mediummay comprise, for example: magnetic storage media such as magnetic disc(such as a floppy disc) or magnetic tape; optical storage media such asoptical disc, optical tape, or machine readable bar code; solid stateelectronic storage devices such as random access memory (RAM), or readonly memory (ROM); or any other physical device or medium which might beemployed to store a computer program. It will be also be understood thatcomputer 100 can be any hardware and/or software combination equivalent,which can execute the steps required by an evaluation method of thepresent invention.

Computer 100 is optionally connected to control an automatedoligonucleotide probe synthesizer, which can either be of a type whichcreates probes of required sequences directly on a substrate 10, or canbe a presynthesizer which synthesizes whole oligonucleotide probes forsubsequent manually controlled or automated linking to substrate 10. Ahybridization chamber 130 is capable of holding an array on substrate 10and a fluid containing target polynucleotide provided from container120, in contact under hybridizing conditions. An array scanner 140 iscapable of observing the binding pattern of such an array and optionallydirectly relaying the results to computer 100. Alternatively, probesynthesizer 114 may be controlled by a user based on sequences output bycomputer 100 such as on display 104 or on a printer (not shown), and theobserved binding pattern data generated by array scanner 140 may beinput to computer 100 by a user (such as through input device 106).

It will be appreciated that in FIG. 6, any one or more of theillustrated components can be remote from the others, and any indicatedconnection can be performed through suitable communication channels forremote components (for example, through a suitable network, such as atelephone network or the internet).

Computer 100 is programmed by the program on medium 110, to execute Asome of the steps of a method of the present invention illustrated inFIG. 7. However, it will be appreciated that some or all of thefollowing steps could be carried out manually by an individual. First,candidate target regions are selected (200). This selection can be basedon any predetermined criteria. For example, candidate regions each of mnucleotides in length and spaced apart by n nucleotides along the targetpolynucleotide can be selected. Such criteria could also, for example,include access to a database of known good target regions for aparticular polynucleotide. Candidate probes which can potentiallyhybridize with the selected respective candidate target regions are thenselected (204). Typically the candidate probes are selected to havesequences which are exact complements of respective target regions. Eachof the candidate probes are then tested (206) on their ability toindividually hybridize with a corresponding target region. This testingstep includes synthesizing the candidate probes at probe synthesizer 114and linking them to a substrate 10 (if the candidate probes are notalready linked to substrate 10 by virtue of being synthesized by in situsynthesis). Note that the candidate probes can be arranged in the formof one or more arrays on substrate 10, each array taking the formdescribed in connection with FIGS. 1 to 3, except each array featurewill typically bear only one candidate sequence. The testing (206)further includes exposing the resulting array to mixtures of targetpolynucleotides from container 120 and maintaining hybridizingconditions while the two are in contact, by means of hybridizationchamber 130. As part of the testing, the array is then removed fromchamber 130 and the resulting binding pattern observed by scanning witharray scanner 140, with the resulting data being received into computer100.

The data, representing a pattern of individual hybridization of thecandidate probes, is then analyzed (208) by computer 100. This analysiscan be based on any suitable algorithm, for example where candidateregions each of m nucleotides in length and spaced apart by nnucleotides along the target polynucleotide were initially selected, theresults for ability of such candidate regions to form individual hybridsversus the position of the target sequence along a targetpolynucleotide, can be analyzed as disclosed in more detail below. Basedon the analysis, an evaluation can be made (210) as to whether betterindividually hybridizing probes might exist. For example, where theanalysis is based on the foregoing ability versus target sequenceposition, if peaks exist in a plot of the data then it may be concludedthat there may be at least one additional candidate target region (atleast partially overlapping the n gap) which might allow for a betterindividually hybridizing probe. Such an additional candidate targetregion is selected and the foregoing steps of selecting a candidatetarget probe, and testing the so-selected candidate target probe, arerepeated using that additional candidate target region. Furtheriterations of the same cycle can be repeated until it is concluded thatit is unlikely that better individually hybridizing probes exist.

At this point, at least one set of at least two of the candidate probeswhich actually hybridized individually with at least a predeterminedefficiency (“successful individually hybridizing candidate probes”) withrespective candidate regions of the same target polynucleotide, is thenselected (212). The one set may include at least two, three or morecandidate probes depending on what efficiency of multidentate binding ofthe target polynucleotide is desired (for example, bidentate, tridentateand the like). Further, multiple sets of such candidate probes may beselected depending upon the results of the individually hybridizingtests. Note that the “predetermined efficiency” can, for example, besome preselected lower limit, or alternatively a preselected number ofthe strongest hybridizing successful individually hybridizing candidateprobes. This selection can, for example, simply be all possiblecombinations of two or more (depending on the efficiency of multidentatebinding desired) successful individually hybridizing candidate probes.Each of the sets is then tested (214) on the ability of the candidateprobes in the set, to form simultaneous hybrids with the respectivecandidate target regions. This testing includes fabricating and testingan array in a similar manner as described above in connection with FIG.6 for testing the ability of candidate probes to individually hybridizeto a respective candidate target regions. However, in the case oftesting for ability of sets to form simultaneous hybrids, each feature16 of the array will carry a mixture of the multiple different targetprobes of the set. In this regard, a portion of such a screening arraymay look and operate in essentially the same manner as described inconnection with FIGS. 3 and 4.

One or more sets of candidate probes which actually formed simultaneoushybrids with respective candidate target regions with at least apredetermined efficiency, may then be selected as a “successfulsimultaneously hybridizing probe set”. Again, the predeterminedefficiency criterion can be any of those criteria mentioned above. Oneor more of such sets can be linked to a substrate 10 at respectivefeatures 16, to form at least part of an array of the present invention.Alternatively, where target probes are intended to be indirectly boundto the substrate through a capture probe, a suitable anti-capturesequence can be selected for the members of a target probe set based onthe capture probe sequence.

EXAMPLE

A specific example of the process of selecting a simultaneouslyhybridizing probe set, will now be described.

Selection of Successful Individually Hybridizing Candidate Probes

Highly sensitive probe sequences specific to the cab, cor47 and sig1genes of Arabidopsis thaliana and a portion of the pbpC gene of E. coliwere determined by two-step iterative refinement. In the first step,every 10th possible 25-mer probe to each target was synthesized on anoligonucleotide array. Arrays were hybridized to rhodamine-6-G-labeledcomplementary RNA (cRNA) derived from each target. Labeled cRNA wasproduced by transcribing a template that placed a T7 RNA polymerasepromoter at the 3′ end of a given gene; rhodamine-6-G (R6G) wasintroduced by adding R6G-CTP (New England Nuclear, Boston, Mass.) to thenucleotide triphosphate mixture.

Hybridizations were performed overnight, at 37° C., in a solutioncontaining 6×SSPE (900 mM sodium chloride/ 60 mM sodium phosphate/ 6 mMEDTA, pH7.5), 0.05% w/v Triton X-100 (Amresco reagent grade, productcode 0694), 100 μg/ml heat-denatured salmon sperm DNA, 1 mg/ml bovineserum albumin, 0.1% w/v sodium dodecyl sulfate and 200 pM cRNA. Arrayswere washed with 0.1×SSPE (1:60 dilution of 6×SSPE) containing 0.005%Triton X-100, at 37° C., for 15 minutes. Washed arrays were dried andread using a confocal laser scanner.

The survey disks yielded “spectra” of probe efficiency: when plotted asa function of probe position in the target coding sequence, thehybridization signals formed a pattern of peaks and valleys. An exampleof such a spectrum for the cor47 gene is shown in FIG. 9.

The peaks observed in each hybridization spectrum were refined by asecond experiment in which every other possible 25-mer probe in eachhybridization peak was synthesized on an oligonucleotide array. Arrayswere hybridized and read as described for the first peak refinementiteration. The results of the first and second design iteration werecombined to pick between 6 and 10 optimized probes to each target; theprobes discovered by this process are listed in Table 1 below.

It should be noted that such empirical probe discovery can be greatlyaided by the use of probe design algorithms. Such algorithms generallyuse predicted thermodynamic properties of candidate probes to a giventarget to predict the approximate locations of peaks of hybridizationefficiency. One such algorithm for selecting individually hybridizingprobes, is disclosed in U.S. patent application Ser. No. 09/021,701,entitled “Methods for Evaluating Oligonucleotide Probe Sequences” filedFeb. 10, 1998 by Karen W. Shannon et al. and owned by the same assigneeas the present application.

TABLE 1 Seq. ID Gene Probe Sequence Probe Name No. cor47GTACCAGTTTCCACTACCATCCCGG cor47-25-0429 001 cor47GAGGATTCACCAGCTGTCACGTCCA cor47-25-0561 002 cor47AGAGGTTACGGATCGTGGATTGTTT cor47-25-0035 003 cor47AGGAGAACAAGATTACTCTGCTAGA cor47-25-0181 004 cor47TGAGGAGAACAAGCCTAGTGTCATC cor47-25-0233 005 cor47AGAGTCTGATGATTAAGCAAAATGG cor47-25-0737 006 sig1TGGTTATGTCTATTGCTCAGCGTTA sig1-25-0477 007 sig1AGGGTGGTCTTATCGGACTTTTGCG sig1-25-0537 008 sig1GTCTTATCGGACTTTTGCGGGGAAT sig1-25-0543 009 sig1GGCTATGAGTAATGTGCGTTTGGTT sig1-25-0457 010 sig1GTGCTTAGTCATGTGGAAGTTGTGC sig1-25-0263 011 sig1GGTCTTCGTCTTGATGATCATAAGT sigI-25-0311 012 sig1GGATTCGACAGGGTGTCTCAAGAGC sig1-25-0621 013 sig1TACATTGCGGATACTCGTTTGGAGA sig1-25-0893 014 cab CACTTGGCGGATCCATGGCACAACAcab-25-0689 015 cab GGATTCTGTGTGCAACAGTCGGCTT cab-25-0629 016 cabGCGCTGTTGGCGTTTGTAGGATTCT cab-25-0611 017 cab GGAGAACTTGGCAACTCACTTGGCGcab-25-0673 018 cab GTGTACCTTATGAGCTTTATGTGTA cab-25-0767 019 cabAACATTGGCGATATTGTTATCCCTT cab-25-0713 020 cab GCTCACTGGATGCCTGGCGAGCCACcab-25-0155 021 cab CATTGCATTTGTTGAGCACCAGAGA Cab-25-0463 022 cabGGTTTGACCCACTTGGACTTGGAGA cab-25-0219 023 cab GTTCCTGGGATTTTGGTACCAGAAGcab-25-0311 024 pbpC GCAGGTTGCTCGTCTGCTTGATCCT pbp1-25-0233 025 pbpCCTCACTATGCAGGTTGCTCGTCTGC pbp1-25-0225 026 pbpCGGTTATTTCCGGTGGCAGCACGCTC pbp1-25-0203 027 pbpCTGCTTGATCCTCACCCCAAAACATT pbp1-25-0247 028 pbpCCTCACTTCGGGACGGGTTATTTCCG pbp1-25-0189 029 pbpCGTGGTCCCGTGAGCAGGTAAAAGAG pbp1-25-0563 030 pbpCATGCGTGTTCGCGGCTGGGTGGGAT pbp1-25-0801 031 pbpCGGCGGTACGTTGCAGGGGATCGGTG pbp1-25-0369 032 pbpCGGATTGCCGTTATATTTGCCCAACG pbp1-25-1131 033

Selection of Successful Simultaneously Hybridizing Probe Set

Effective binary probe combinations (target probe sets) can then bedetermined by combinatorial means, using an array as the measurementdevice. First, test features are designed on the basis of the optimalprobes previously determined for that target. For example, for thetarget cor47 (see Table 1), the binary probe combinations to be screenedare (in terms of Sequence ID Numbers) 001&002, 001&003, 001&004,001&005, 001&006, 002&003,002&004, 002&005, 002&006, 003&004, 003&005,003& 006, 004&005, 004&006 and 005&006. This example is easily extendedto the probe lists for each target considered in Table 1. In general, ifprobe optimization has defined N effective probes against a giventarget, then the number of binary probe combinations B to be screened isgiven by $B = {\frac{N( {N - 1} )}{2}.}$

Screening is performed on an array that compares the hybridizationefficiencies of the binary features to the hybridization efficiencies ofthe N original optimized probes. If each feature is repeated M times,then F, the total number of features needed, is given by $\begin{matrix}{F = {M( {B + N} )}} \\{= {M\lbrack {\frac{N( {N - 1} )}{2} + N} \rbrack}} \\{= \frac{{MN}( {N + 1} )}{2}}\end{matrix}$

For the example of cor47, if M=3 (a minimal value for an optimizationexperiment), then F is 63. It is easy to see that the number of requiredfeatures per gene is significant even if the best probes are pickedprior to initiating optimization of binary combinations. However,pre-picking the best probes is much more efficient than testing allprobe combinations. For example, testing all possible binarycombinations of every other probe against cor47 (i.e. the set requiredto guarantee that the optimal combinations are all examined) requires anN of 369 (i.e. every other probe, starting at 1 and ending at 737). IfM=3, then F is 204,795. This is almost twice the number of featuresavailable on the highest density arrays made by current commercialmeans, and is inconveniently large.

The above discussion can be generalized to features that combine kprobes. In the general case, K, the number of unique combinations of kprobes that can be produced from a set of N unique probes, is given by$K = {\frac{N!}{{k!}{( {N - k} )!}}.}$

The total number of features F required to compare all unique mixturesof k probes to the original set of N probes is given by$F = {{M\lbrack {\frac{N!}{{k!}{( {N - k} )!}} + N} \rbrack}.}$

Again, the example of cor47 is instructive. If 6 pre-optimized probesare used in unique combinations of 3 (k=3) and measurements are repeated3 times (M=3), F is 78. If the probes are not pre-optimized (N=369),then F is 24,918,939. Clearly, the use of pre-optimized probes is muchmore efficient.

During the initial screen of combination probe features, equimolarmixtures of probes are used. Based on the results of the initial screen,the best combinations are chosen for further optimization by systematicvariation of the mole fraction of each component probe.

Determination of Optimum Stoichiometry of Probe Set

It would initially appear that the probability of multidentate bindingof a target by a target probe set would be a maximum when all memberprobes are present in equal numbers. However, several effects can changethe optimal mixture to one in which probes are not equally represented.The probes may not mix ideally. Binding of target to one probe maycreate a radial or angular “shadow”, rendering other probes inactive.Some probes are capable of binding to more than one region of sometargets. For these reasons, the relative concentrations of target probesin a set used to construct a multiprobe feature, should be optimizedempirically. This can be accomplished by, for example, using someversion of grid search (that is, test different probe set mixturescontaining different relative concentrations of the member targetprobes, in an organized fashion, then use the surface shape to find theoptimum), or via a statistical experimental design, such as a factorialor Taguchi design. Note that tests of a probe set at multiple relevantconcentrations will generally be done under the same set of conditions(for example, same concentration of components, and same time andtemperature). Generally, a grid search will be more efficient for binaryor trinary of target probe sets. Statistical experimental designs aremore efficient for combinations of many probes. Both approaches can beapplied under the guidance of commercially available software packagesany of which could be run by computer 100. In this event, it will beappreciated that computer 100 can also select the various concentrationsof a selection successful simultaneously hybridizing probe set fortesting, and receive and analyze the test results to select particularrelative concentrations to be linked to a substrate at a predeterminedfeature, during fabrication of an array of the present invention.

The disclosed invention then, can provide high sensitivity for a targetby using array probe features that contain a mixture of particulartarget probes. Such array features combine the affinity of long probesand the specificity of shorter probes.

Various modifications to the embodiments of the invention describedabove are, of course, possible. Accordingly, the present invention isnot limited to the particular embodiments described in detail above.

33 1 25 DNA Arabidopsis thaliana 1 gtaccagttt ccactaccat cccgg 25 2 25DNA Arabidopsis thaliana 2 gaggattcac cagctgtcac gtcca 25 3 25 DNAArabidopsis thaliana 3 agaggttacg gatcgtggat tgttt 25 4 25 DNAArabidopsis thaliana 4 aggagaacaa gattactctg ctaga 25 5 25 DNAArabidopsis thaliana 5 tgaggagaac aagcctagtg tcatc 25 6 25 DNAArabidopsis thaliana 6 agagtctgat gattaagcaa aatgg 25 7 25 DNAArabidopsis thaliana 7 tggttatgtc tattgctcag cgtta 25 8 25 DNAArabidopsis thaliana 8 agggtggtct tatcggactt ttgcg 25 9 25 DNAArabidopsis thaliana 9 gtcttatcgg acttttgcgg ggaat 25 10 25 DNAArabidopsis thaliana 10 ggctatgagt aatgtgcgtt tggtt 25 11 25 DNAArabidopsis thaliana 11 gtgcttagtc atgtggaagt tgtgc 25 12 25 DNAArabidopsis thaliana 12 ggtcttcgtc ttgatgatca taagt 25 13 25 DNAArabidopsis thaliana 13 ggattcgaca gggtgtctca agagc 25 14 25 DNAArabidopsis thaliana 14 tacattgcgg atactcgttt ggaga 25 15 25 DNAArabidopsis thaliana 15 cacttggcgg atccatggca caaca 25 16 25 DNAArabidopsis thaliana 16 ggattctgtg tgcaacagtc ggctt 25 17 25 DNAArabidopsis thaliana 17 gcgctgttgg cgtttgtagg attct 25 18 25 DNAArabidopsis thaliana 18 ggagaacttg gcaactcact tggcg 25 19 25 DNAArabidopsis thaliana 19 gtgtacctta tgagctttat gtgta 25 20 25 DNAArabidopsis thaliana 20 aacattggcg atattgttat ccctt 25 21 25 DNAArabidopsis thaliana 21 gctcactgga tgcctggcga gccac 25 22 25 DNAArabidopsis thaliana 22 cattgcattt gttgagcacc agaga 25 23 25 DNAArabidopsis thaliana 23 ggtttgaccc acttggactt ggaga 25 24 25 DNAArabidopsis thaliana 24 gttcctggga ttttggtacc agaag 25 25 25 DNAEscherichia coli 25 gcaggttgct cgtctgcttg atcct 25 26 25 DNA Escherichiacoli 26 ctcactatgc aggttgctcg tctgc 25 27 25 DNA Escherichia coli 27ggttatttcc ggtggcagca cgctc 25 28 25 DNA Escherichia coli 28 tgcttgatcctcaccccaaa acatt 25 29 25 DNA Escherichia coli 29 ctcacttcgg gacgggttatttccg 25 30 25 DNA Escherichia coli 30 gtggtcccgt gagcaggtaa aagag 25 3125 DNA Escherichia coli 31 atgcgtgttc gcggctgggt gggat 25 32 25 DNAEscherichia coli 32 ggcggtacgt tgcaggggat cggtg 25 33 25 DNA Escherichiacoli 33 ggattgccgt tatatttgcc caacg 25

What is claimed is:
 1. A method of evaluating for the presence ofmultiple different target polynucleotides in a same sample, using anaddressable array of at least one hundred features having differentpolynucleotidc probes linked to a substrate, the method comprising: (a)exposing the same sample to the array and different target probe setseach having different polynucleotide target probes, such that each ofthe different target polynucleotides which may be present will bind to acorresponding predetennined feature of the array through multiple targetprobes of a corresponding target probe set by forming at respectivetarget regions on a target molecule, simultaneous hybrids withanti-target regions of the multiple target probes of Me correspondingtarget probe set; and (b) observing a binding pattern on the array andevaluating the presence of the target polynucleotide based on theobserved binding pattern wherein: (i) the target probes are linked tothe substrate at the predetermined features prior to exposing thesample; or (ii) the target probes are not linked to the substrate priorto exposing the sample, and also include anti-capture regions; and thesample is also exposed to a set of capture probes linked to thesubstrate as part of the array, which have capture regions which willhybridize with respective anti-capture regions; so that each of thedifferent target polynucleotides which may be present will eachindirectly bind to the corresponding predetermined feature for thattarget with the target probes of the corresponding target probe set alsohaving their anti-capture regions hybridizing with the capture regionsof the capture probes.
 2. A method according to claim 1 wherein therespective target regions on each of the different targets are ofdifferent sequence, and each set of target probes has at least twotarget probes with different sequence anti-target regions.
 3. A methodaccording to claim 2 wherein the target regions differ in sequence fromone another by at least two nucleotides, and the anti-target regions ofeach set differ from one another by at least two nucleotides.
 4. Amethod according to claim 2 wherein the target regions differ insequence from one another by at least four nucleotides, and theanti-target regions of each set differ from one another by at least fournucleotides.
 5. A method according to claim 2 wherein the target probesare directly linked to the substrate at the predetermined features.
 6. Amethod according to claim 1 wherein each set of target probes has atleast three polynucleotide target probes with different sequenceanti-target regions.
 7. A method according to claim 1 wherein the targetprobes are linked to the substrate at the predetermined feature prior toexposing the sample.
 8. A method according to claim 1 wherein: thetarget probes are not linked to the substrate, and also includeanti-capture regions; and the predetermined features of the array eachincludes a set of capture probes linked thereto, and which have captureregions which will hybridize with respective anti-capture regions; sothat multiple molecules of each target polynucleotide which may bepresent will each indirectly bind to the corresponding predeterminedfeature by the anti-target regions of the multiple target probes formingthe simultaneous hybrids with the respective target regions and by theanti-capture regions hybridizing with the capture regions of the captureprobes.
 9. A method according to claim 8 wherein the target regions areof different sequence, and the sets of target probes each has at leasttwo target probes with different sequence anti-target regions.
 10. Amethod according to claim 9 wherein the anti-capture regions of the setare of the same sequence.
 11. A method according to claim 8 wherein theanti-capture regions of target probes in a set are of differentsequence.
 12. A method according to claim 1 wherein, based on theobserved binding pattern, target polynucleotide is determined to havebeen present in the sample.
 13. A method according to claim 1 whereinthe target probes are directly linked to the substrate at thepredetermined features.
 14. An apparatus for evaluating for the presenceof multiple different target polynucleotides in a same sample,comprising: (a) an addressable array of at least one hundred featureshaving different polynucleotide probes linked to the substrate: (b)different sets of polynucleotide target probes each having differentpolynucleotide target probes, such that each of the different targetpolynucleotides which may be present in a sample exposed to the arraywill bind to a corresponding predetermined feature of the array throughmultiple different target probes of a corresponding target probe set byforming at respective target regions on a target molecule, simultaneoushybrids with anti-target regions of the multiple target probes of thecorresponding target probe set; wherein the target regions of each setare of different sequence, and each of the sets of target probes has atleast two target probes with different sequence anti-target regions: andwherein: (i) the target probes are linked to the substrate at thepredetermined features; or (ii) the target probes also includeanti-capture regions; and the apparatus additionally comprise a set ofcapture probes linked to the substrate as part of the array, which havecapture regions which will hybridize with respective anti-captureregions; so that each of the different target polynucleotides which maybe present will each indirectly bind to the corresponding predeterminedfeature for that target with the target probes of the correspondingtarget probe set also having their anti-capture regions hybridizing withthe capture regions of the capture probes.
 15. An apparatus according toclaim 14 wherein the anti-target regions of the multiple target probesof the different sets are all of different sequence.
 16. An apparatusaccording to claim 15 wherein the target probes are directly linked tothe substrate at the predetermined features.
 17. An apparatus methodaccording to claim 14 wherein a set of target probes has at least threetarget probes with different sequence anti-target regions.
 18. Anapparatus according to claim 14 wherein each of the sets of targetprobes are linked to the substrate at the corresponding predeterminedfeature.
 19. An apparatus according to claim 14 wherein: each set oftarget probes of the set are not linked to the substrate, and alsoinclude anti-capture regions; each predetermined feature of the arrayincludes capture probes linked to the substrate which have captureregions which will hybridize with the anti-capture regions of acorresponding target probe set; so that multiple molecules of eachtarget polynucleotide which may be present will each indirectly bind tothe corresponding predetermined feature by the anti-target regions ofthe multiple target probes forming the simultaneous hybrids with therespective target regions and by the anti-capture regions hybridizingwith the capture regions of the capture probes.
 20. An apparatusaccording to claim 19 wherein the target regions of each target are ofdifferent sequence, and each set of target probes has at least twotarget probes with different sequence anti-target regions.
 21. A methodaccording to claim 20 wherein the anti-capture regions of a set are ofthe same sequence.
 22. An apparatus according to claim 19 wherein theanti-capture regions of target probes in a set are of differentsequence.
 23. An apparatus according to claim 14 wherein the targetregions differ in sequence from one another by at least two nucleotides,and the anti-target regions of each target probe set differ from oneanother by at least two nucleotides.
 24. An apparatus according to claim14 wherein the target regions differ in sequence from one another by atleast four nucleotides, and the anti-target regions of each target probeset differ from one another by at least four nucleotides.
 25. Anapparatus according to claim 14 wherein the target probes are directlylinked to the substrate at the predetermined features.